Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) is the most severe pandemic disease in modern history and remains a major threat to humans. With HIV/AIDS prevention, diagnosis and treatment, the morbidity and mortality of AIDS decreased significantly. However, AIDS remains an incurable, chronic infection due to the multiple HIV latent cells in a patient.
HIV infection of human cells can be divided into active infection and latent infection. In most human cells, HIV infection is active infection; however, in rare human cells, latent infection can occur at very early stage. These very small numbers of latently infected cells are called HIV reservoirs and they are located mainly in brain, peripheral blood, and lymphoid tissue. The HIV reservoir cells include resting memory T cells in lymph nodes; astrocytes, microglia, and microphages in brain; and resting memory T cells and monocytes in peripheral blood.
To date, the mechanism of the formation of the reservoir and activation of the latent cells remains largely unknown; however, in reservoir cells HIV provirus may be silenced by multiple factors, including integration site, chromatin status, accessibility of transcription factors, and ribonucleic acid (RNA) interference. In the reservoir cell sites antiviral drugs penetration is often low. Moreover, even under antiretroviral therapy (ART), about 30 to 50% of AIDS patients eventually develop HIV-associated neurological disorders (HAND), which are cognitive, motor and/or behavioral impairments caused by HIV infection in human brain. HAND can further be grouped into asymptomatic neurocognitive impairment (ANI), minor neurocognitive disorder (MND) and the most severe HIV-associated dementia (HAD).
The mechanism of HAND remains to be elucidated; however, HAND is tightly correlated with HIV infection of astrocytes, microglia and macrophages in human brains. Neurons are believed to be resistant to HIV infection. However, the neurotoxic products released from HIV infected brain cells seriously dysregulates neuronal function and homeostasis.
Astrocytes are very important supporting cells in human central nervous system and they play critical roles in physiological and pathological conditions. Their functions include being a critical component of Blood Brain Barrier, forming tripartite synapses; being structural scaffold, releasing and up taking neurotransmitters and providing energy substrates to neurons. Moreover, in some pathological conditions, astrocytes' ability to maintain homeostasis is disrupted. HIV and HIV proteins impair astrocytes' ability to maintain homeostasis.
RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is derived from the adaptive immune system from bacteria. Cas9 can be bioengineered for better nucleus localization and mammalian cell expression. The original two RNA (crRNA and tracRNA) can be converted into a single guided RNA (gRNA). Cas9 has two nuclease activity domains named HNH and RuvC. Each of these two domains can cleave a DNA strand directed by a gRNA complementary to the target DNA sequence. The prerequisite to be a target sequence is the presence of a NGG sequence (protospacer adjacent motifs, PAM) downstream the target site and the target sequence is generally 20 nucleotides. In the nucleus, Cas9, gRNA, and target DNA form a complex and HNH and RuvC domains each cleave a DNA strand. The double strand breaks are consequently repaired mainly by two approaches: non-homologous end-joining (NHEJ) when a template is absent or homology directed repair (HDR) when a homogenous repair template is present. NHEJ usually results in insertion or deletion (indel), while HDR results in correct repair as directed by the template.
The ultimate cure for HIV/AIDS would be the removal or disruption of integrated HIV provirus in latently infected cells or the complete elimination of these latent cells. However, gene therapy for HIV/AIDS has progressed very slowly.